AIM:To observe the effect of folic acid(FA)on C2C12 myoblast proliferation and differentia-tion,and to explore its mechanism.METHODS:During the proliferation stage,C2C12 myoblasts were treated with vari-ous concentrations of FA(0,2.5,5,10 and 20 μmol/L).The cell status was observed under a microscope,cell viability was detected using the MTT method,and cell proliferation was assessed using the EdU method.In the differentiation stage,C2C12 cells were divided into control(Ctrl)group(0 μmol/L FA)and FA group(10 μmol/L FA).On day 2 or 4 of differentiation,immunofluorescence staining and Western blot were employed to detect the expression of myoblast differen-tiation-related proteins,myoblast determination protein 1(MyoD),myogenin(MyoG)and myosin heavy chain(MyHC).The myotubule formation in each group was analyzed.On day 4 of differentiation,C2C12 cells were treated with FA for 0,1,3 and 6 h,and the protein levels of p-JNK,JNK,p-p38 MAPK and p38 MAPK at each time point were detected by Western blot.Additionally,C2C12 cells after 4-day differentiation were divided into Ctrl group,FA group,FA+ SP600125(specific inhibitor of JNK)group,and FA+SB203580(specific inhibitor of p38)group.The cells in FA+ SP600125 and FA+SB203580 groups were treated with 10 μmol/L SP600125 or SB203580 for 1 h,followed by treatment with 10 μmol/L FA for 24 h.The cells in FA group were treated with 10 μmol/L FA for 24 h,while the cells in Ctrl group were left untreated.The protein levels of p-JNK,JNK,p-p38 MAPK,p38 MAPK and MyHC were detected by Western blot.RESULTS:(1)Compared with 0 μmol/L FA group,the number of the cells in other concentration groups in-creased,cell viability was raised(P＜0.05 or P＜0.01),and the rate of EdU positive cells increased(P＜0.05).(2)Com-pared with Ctrl group,the expression levels of MyoD,MyoG and MyHC in FA group were increased(P＜0.05),and the myotube fusion index was raised(P＜0.05 or P＜0.01).(3)Compared with 0 h group,the ratios of p-JNK/JNK and p-p38 MAPK/p38 MAPK were elevated after FA treatment for 1,3 and 6 h(P＜0.05 or P＜0.01),and showed a trend of gradual increase with the extension of treatment time.(4)After FA treatment,the ratios of p-JNK/JNK and p-p38 MAPK/p38 MAPK,and the expression of MyHC were elevated(P＜0.01).Treatment with SP600125 decreased the ratio of p-JNK/JNK and the expression of MyHC(P＜0.05),while SB203580 intervention cut down the ratio of p-p38 MAPK/p38 MAPK and the expression of MyHC(P＜0.05 or P＜0.01).CONCLUSION:Folic acid can promote the differentiation of C2C12 myoblasts by activating the JNK/p38 MAPK signaling pathway.

Objective:To analyze the mutation of T-cell and B-cell epitopes derived from HBV PreS-S protein in occult hepatitis B virus (OHBV) and investigate the biological mechanisms of occult hepatitis B virus infection (OBI) and HBsAb positive OBI.Methods:The PreS-S region of OBI samples were amplified by nested PCR, the products were sequenced and HBV genotypes were determined. The mutations of T-cell and B-cell epitopes derived from HBV PreS-S protein were analyzed and compared among groups of HBV genotypes and the presence of HBsAb. The affinity of the high frequency of T-cell epitope substitutions were analyzed by SYF PEITHI, the changes of antigenic characteristics of high frequency of B-cell epitope substitutions were analyzed by Ab Designer, Expasy ProtParam tool, Epitope Prediction and Analysis Tools.Results:The PreS-S region of HBV was amplified in 21 samples, including 4 HBsAb+ OBI B, 6 HBsAb-OBI B, 6 HBsAb+ OBI C, 5 HBsAb-OBI C. The mutation rates in PreS-S region of OBI were significantly higher than wild type HBV strains(OBI Bvs. WT B: 2.64%: 0.66%, P<0.001; OBI Cvs. WT C: 3.67%: 1.19%, P<0.001). The mutation rates of the immunoreactive area were significantly higher than non-immunoreactive area in OBI (OBI B: 3.57%: 1.86%, P=0.005; OBI C: 4.78%: 2.65%, P<0.001). The mutation rates of the immunoreactive and non-immunoreactive area in OBI C were higher than OBI B, but there was no statistically significant difference (immunoreactive area: 4.78%: 3.57%, P=0.107; non-immunoreactive area: 2.65%: 1.86%, P=0.142). The mutation rates of T-cell and B-cell epitopes of HBsAb-OBI were higher than HBsAb+ OBI, although there was no significant difference (HBsAb-OBI Bvs. HBsAb+ OBI B: 4.17∶3.01, P=0.303; HBsAb-OBI Cvs. HBsAb+ OBI C: 5.65∶4.26, P=0.207). The affinity analysis of 4 high frequency T-cell epitope substitutions, including T47A/K, S174N, L175S, V177A, showed that the changes of affinity of most mutation sites were not obvious; the antigenicity analysis of 3 high frequency B-cell epitope substitutions, including G73S, K122R, I126M/T, did not show noticeable changes and the hydrophilicity, surface accessibility of some mutation sites were even better than wild strain. Conclusions:The mutation rates in PreS-S region of OBI were significantly higher than wild type HBV strains. The mutation rates of the immunoreactive area were higher than non-immunoreactive area in OBI. The variant activity of OBI C was higher than OBI B. The mutations of OBI might occur randomly and were not selected by antibody pressure. Single epitope and multi-epitopes combinational mutations might be a reason for OBI.

Objective To monitor the antifungal resistance of clinical isolates of Candida spp.in the East China region.Methods MALDI-TOF MS or molecular methods were used to re-identify the strains collected from January 2018 to December 2022.Antifungal susceptibility testing was performed using the broth microdilution method.The susceptibility test results were interpreted according to the breakpoints of 2022 Clinical and Laboratory Standards Institute(CLSI)documents M27 M44s-Ed3 and M57s-Ed4.Results A total of 3 026 strains of Candida were collected,65.33％of which were isolated from sterile body sites,mainly from blood(38.86％)and pleural effusion/ascites(10.21％).The predominant species of Candida were Candida albicans(44.51％),followed by Candida parapsilosis complex(19.46％),Candida tropicalis(13.98％),Candida glabrata(10.34％),and other Candida species(0.79％).Candida albicans showed overall high susceptibility rates to the 10 antifungal drugs tested(the lowest rate being 93.62％).Only 2.97％of the strains showed dose-dependent susceptibility(SDD)to fluconazole.Candida parapsilosis complex had a SDD rate of 2.61％and a resistance rate of 9.42％to fluconazole,and susceptibility rates above 90％to other drugs.Candida glabrata had a SDD rate of 92.01％and a resistance rate of 7.99％to fluconazole,resistance rates of 32.27％and 48.24％to posaconazole and voriconazole non-wild-type strains(NWT),respectively,and susceptibility rates above 90％to other drugs.Candida tropicalis had resistance rates of 29.55％and 26.24％to fluconazole and voriconazole,respectively,resistance rates of 76.60％and 21.99％to posaconazole and echinocandins non-wild-type strains(NWT),and a resistance rate of 2.36％to echinocandins.Conclusions The prevalence and species distribution of Candida spp.in the East China region are consistent with previous domestic and international reports.Candida glabrata exhibits certain degree of resistance to fluconazole,while Candida tropicalis demonstrates higher resistance to triazole drugs.Additionally,echinocandins resistance has emerged in Candida albicans,Candida glabrata,Candida tropicalis,and Candida parapsilosis.

Objective:To explore the characteristics of brain dynamic activity in patients with schizophrenia by using functional magnetic resonance imaging(fMRI).Methods:Forty-three patients with schizophrenia and 31 normal controls were recruited and under fMRI scanning.The Positive and Negative Symptom Scale(PANS)was used to assess the severity of clinical symptoms.The DPABI software were used to compute dReHo and dFC.Granger causality analysis was used to calculate the effective connectivity between the significant brain regions of dReHo and the whole brain.Two sample t-test was performed to compare the difference of dReHo and dFC be-tween patients with schizophrenia and normal controls.Results:The dReHo of left postcentral gyrus(LPG)(P＜0.01,cluster-level FWE corrected)in patients with schizophrenia was decreased.The Dfc was increased between left postcentral and left middle frontal gyrus,left superior medial frontal gyrus,right calcarine,left medial cingulum gyrus,right supplementary motor area(P＜0.01,uncorrected).Compared with normal controls,patients with schiz-ophrenia showed decreased effective connectivity from LPG to right putamen.Conclusion:It suggests that the ab-normal functional activity of the postcentral gyrus mightcontribute to the neural physiopathology in patients with schizophrenia.

Objective:To clarify whether CLDN5 is involved in the pathogenesis of sensitive skin (SS) through inflammation.Methods:From January 2018 to March 2020, in the Dermatology Laboratory of the First Affiliated Hospital of Kunming Medical University, facial tissues were collected from 5 patients diagnosed with sensitive skin and 5 cases of normal facial skin. The organizational experiment was divided into two groups: SS group and normal skin (NS) group. The cell experiment was divided into two groups: sh-CLDN5 group in which CLDN5 was knocked down and sh-NC group with normal expression of CLDN5. After paraffin embedding and sectioning, immunohistochemical staining was performed to determine the expression of claudin-5 in sensitive skin and normal skin. In cell experiments, lentivirus shRNA was transfected into HaCaT cells. Three cases in sh-CLDN5 group and three cases in sh-NC group were collected for transcriptome sequencing, and differentially expressed pro-inflammatory factor mRNA was screened from the sequencing results. Western blot or ELISA was used to verify the protein level expression of differentially expressed pro-inflammatory cytokines mRNA in HaCaT cells, and Western blot was used to detect the expression of key proteins in MAPK pathway and NF-κB in the sh-CLDN5 group and sh-NC group.Results:Immunohistochemical staining showed that claudin-5 was localized in the interstitial cells of the normal skin granular layer. Patients with sensitive skin had thinner epidermis than normal skin and significantly reduced claudin-5 expression. In HaCaT cells, the expression of pro-inflammatory cytokine IL-23A was increased, and IL-8 was elevated in the sh-CLDN5 group [(272.91±30.25) pg/ml, n=3] compared with sh-NC group [(55.58±7.07) pg/ml, n=3] ( P<0.01). There was no statistically significant difference in secretion volume between sh-NC group [(8.04±1.34) pg/ml, n=3] and sh-CLDN5 group [(12.15±3.07) pg/ml, n=3]. The expression of P-JNK, p-ERK, and P-p38 in the MAPK pathway was significantly increased ( P<0.01). Conclusions:CLDN5 is an important gene involved in the pathogenesis of SS. CLDN5 upregulates IL-23A and IL-8 in HaCaT and activates the MAPK pathway, which is involved in the process of early SS inflammation.

Purpose: Whether a dilated intrahepatic bile duct (IHBD) has any effect on the prognosis of choledochal cyst (CC) remains controversial. We aimed to summarize the clinical characteristics and prognosis of CC with IHBD dilatation. Methods: One hundred ninety-two children diagnosed with CC were identified, including 127 without IHBD dilatation (group A) and 65 with IHBD dilatation (group B). A retrospective analysis was performed to explore the clinical characteristics and prognosis of CC with IHBD dilatation based on clinical indices, symptoms, and complications. Results: Compared with group A, incidences of jaundice and fever were higher in group B (P = 0.010 and P = 0.033). Preoperative total bilirubin, direct bilirubin, and indirect bilirubin were increased in group B compared to group A (P = 0.005, P < 0.001, and P = 0.014), as were preoperative ALT, AST, γ-GT, and total bile acid (P = 0.006, P = 0.025, P < 0.001, and P = 0.024). The risk of liver fibrosis or cirrhosis was significantly increased for group B compared with group A (P = 0.012) and also occurred earlier in group B (P = 0.006). In the dilated IHBDs, 95.4% (62 of 65) recovered to normal, and more than half of dilated IHBDs (37 of 65) recovered to normal in 1 week. Conclusion Most IHBDs can recover to normal postoperatively in a short time, and proactive treatment is recommended for CC patients with IHBD dilatation for significant abnormal liver functions.

Hip fracture is considered as the most severe osteoporotic fracture characterized by high disability and mortality in the elderly. Improved surgical techniques and multidisciplinary team play an active role in alleviating prognosis, which places higher demands on perioperative nursing. Dysfunction, complications, and secondary impact of anaesthesia and surgery add more difficulties to clinical nursing. Besides, there still lack clinical practices in perioperative nursing for elderly patients with hip fracture in China. In this context, led by the Orthopedic Nursing Committee of Chinese Nursing Association, the Expert consensus on clinical practice in perioperative nursing for elderly patients with hip fracture ( version 2023) is developed based on the evidence-based medicine. This consensus provides 11 recommendations on elderly patients with hip fracture from aspects of perioperative health education, condition monitoring and inspection, complication risk assessment and prevention, and rehabilitation, in order to provide guiding advices for clinical practice, improve the quality of nursing and ameliorate the prognosis of elderly patients with hip fracture.

Objective:To investigate the diagnostic accuracy of serological indicators and evaluate the diagnostic value of a new established combined serological model on identifying the minimal hepatic encephalopathy (MHE) in patients with compensated cirrhosis.Methods:This prospective multicenter study enrolled 263 compensated cirrhotic patients from 23 hospitals in 15 provinces, autonomous regions and municipalities of China between October 2021 and August 2022. Clinical data and laboratory test results were collected, and the model for end-stage liver disease (MELD) score was calculated. Ammonia level was corrected to the upper limit of normal (AMM-ULN) by the baseline blood ammonia measurements/upper limit of the normal reference value. MHE was diagnosed by combined abnormal number connection test-A and abnormal digit symbol test as suggested by Guidelines on the management of hepatic encephalopathy in cirrhosis. The patients were randomly divided (7∶3) into training set ( n=185) and validation set ( n=78) based on caret package of R language. Logistic regression was used to establish a combined model of MHE diagnosis. The diagnostic performance was evaluated by the area under the curve (AUC) of receiver operating characteristic curve, Hosmer-Lemeshow test and calibration curve. The internal verification was carried out by the Bootstrap method ( n=200). AUC comparisons were achieved using the Delong test. Results:In the training set, prevalence of MHE was 37.8% (70/185). There were statistically significant differences in AMM-ULN, albumin, platelet, alkaline phosphatase, international normalized ratio, MELD score and education between non-MHE group and MHE group (all P<0.05). Multivariate Logistic regression analysis showed that AMM-ULN [odds ratio ( OR)=1.78, 95% confidence interval ( CI) 1.05-3.14, P=0.038] and MELD score ( OR=1.11, 95% CI 1.04-1.20, P=0.002) were independent risk factors for MHE, and the AUC for predicting MHE were 0.663, 0.625, respectively. Compared with the use of blood AMM-ULN and MELD score alone, the AUC of the combined model of AMM-ULN, MELD score and education exhibited better predictive performance in determining the presence of MHE was 0.755, the specificity and sensitivity was 85.2% and 55.7%, respectively. Hosmer-Lemeshow test and calibration curve showed that the model had good calibration ( P=0.733). The AUC for internal validation of the combined model for diagnosing MHE was 0.752. In the validation set, the AUC of the combined model for diagnosing MHE was 0.794, and Hosmer-Lemeshow test showed good calibration ( P=0.841). Conclusion:Use of the combined model including AMM-ULN, MELD score and education could improve the predictive efficiency of MHE among patients with compensated cirrhosis.

Our recent studies for nonnucleoside reverse transcriptase inhibitors identified a highly potent compound JK-4b against WT HIV-1 (EC₅₀ = 1.0 nmol/L), but the poor metabolic stability in human liver microsomes (t _1/2 = 14.6 min) and insufficient selectivity (SI = 2059) with high cytotoxicity (CC₅₀ = 2.08 μmol/L) remained major issues associated with JK-4b. The present efforts were devoted to the introduction of fluorine into the biphenyl ring of JK-4b, leading to the discovery of a novel series of fluorine-substituted NH₂-biphenyl-diarylpyrimidines with noticeable inhibitory activity toward WT HIV-1 strain (EC₅₀ = 1.8-349 nmol/L). The best compound 5t in this collection (EC₅₀ = 1.8 nmol/L, CC₅₀ = 117 μmol/L) was 32-fold in selectivity (SI = 66,443) compared to JK-4b and showed remarkable potency toward clinically multiple mutant strains, such as L100I, K103N, E138K, and Y181C. The metabolic stability of 5t was also significantly improved (t _1/2 = 74.52 min), approximately 5-fold higher than JK-4b in human liver microsomes (t _1/2 = 14.6 min). Also, 5t possessed good stability in both human and monkey plasma. No significant in vitro inhibition effect toward CYP enzyme and hERG was observed. The single-dose acute toxicity test did not induce mice death or obvious pathological damage. These findings pave the way for further development of 5t as a drug candidate.

Objective:To discuss the molecular association pattern of Shenling Baizhu Powder for type 2 diabetes (T2DM) and ulcerative colitis through network pharmacology method based on the theory of "treating different diseases with same method" in TCM.Methods:The TCMSP and ETCM Chinese medicine chemical composition databases were used to obtain the chemical composition and predict the targets of Shenling Baizhu Powder. The OMIM, GeneCards, DrugBank and TTD disease databases were used to obtain the disease targets of T2DM and ulcerative colitis. The intersection targets of chemical composition of Shenling Baizhu Powder, T2DM and ulcerative colitis were obtained by Bioinformatics platform. Cytoscape 3.8.2 software was used to map the "Chinese materia medica-component-target" network and "effective component- intersection target" network. The GO enrichment analysis and KEGG pathway analysis of the intersection targets were performed with the STRING platform. The intersection target protein-protein interaction network was constructed by STRING database, and core targets were obtained using the CytoNCA plugin of Cytoscape 3.8.2 software. AutodockTools software was applied to validate the molecular docking between the core chemical components and the core targets of Shenling Baizhu Powder.Results:Totally 176 chemical components of Shenling Baizhu Powder, 226 corresponding targets, 11 478 T2DM targets, 4 857 Ulcerative targets of ulcerative colitis, and 162 intersection targets of medicinal chemistry components and diseases were obtained. 1 789 related biological processes, 163 molecular functions, 92 cell constituents, and 192 signaling pathways were obtained by GO enrichment analysis and KEGG pathway analysis. The signaling pathways were mainly about AGE-RAGE, TNF, IL-17,MAPK, PI3K-Akt signaling pathways, etc. The core components of Shenling Baizhu Powder were mainly sitosterol, luteolin, kaempferol, naringenin, beta-carotene, etc. The core targets were EGFR, ALB, IL1B, CASP3, ESR1, VEGFA, PTGS2, TNF, IL6, MYC, AKT 1, JUN, TP53, etc. The core chemical components had tight correlation with the core targets by the molecular docking.Conclusions:By acting on core targets such as TNF, IL1B, IL6, AKT1, VRGFA, PTGS2, MYC, JUN, TP53, and regulating IL-17 signaling pathway, TNF signaling pathway, MAPK signaling pathway, AGE-RAGE signaling pathway, etc., Shenling Baizhu Powder improves tissue inflammation damage, mucosal barrier damage, immune regulation imbalance, intestinal flora dysbiosis, insulin resistance, apoptosis, oxidative stress and other biological processes. Therefore Shenling Baizhu Powder can treat T2DM and ulcerative colitis with the same method.